We have developed a simplified protocol for the isolation and purification of human C2. Interestingly, co-isolating with C2 by this procedure is a previously unidentified protein similar to C2 but with unique properties. This protein has no C2 hemolytic activity but may cross-react antigenically with C2. It has a chain structure on reduced and non-reduced SDS-PAGE gels similar to C2 (MW 105,000) but is slightly heavier (MW 120,000). On agarose electrophoresis the 120 KD protein migrates anodal to C2 and elutes from DEAE Sephacel at slightly higher ionic strength implying a more acidic pI. Similar iodine incorporation was seen with both the 120 KD protein and C2; 125I-incorporation with N- chloro-benzenesulfonamide yielded 10X the specific radioactivity as with Bolton-Hunter reagent. The 120 KD protein is recovered in the same polyethylene glycol fraction as C2, binds to C4b/iC4 in the presence or absence of C2 and is recovered in somewhat greater yield than C2, about 2mg/100ml EDTA plasma. Preliminary evidence suggests that this 120 KD protein binds to C4b/iC4 and exhibits cofactor activity for enhancement of C2 lytic function. Between two and 10 fold potentiation of C2 lytic activity was observed by prior treatment of target EAC14b cells with up to 9 Mug/ml 120 KD protein. By a number of immunochemical techniques (Western blot, immunoelectrophoresis and double diffusion analysis), the 120 KD protein has been shown not to be antigenically related to factor B, C6, C7, C4BP or to any of eleven other serum proteins tested.